It is known that ADP (adenosine-5xe2x80x2-diphosphate) plays a pivotal role in terms of platelet function by causing adhesion, degranulation, shape change and aggregation of platelets via interaction with cell surface P2 receptors. It is the P2YADP receptor (formerly known as P2T) that is primarily involved in mediating platelet aggregation, which is an as yet uncloned G-protein linked receptor. The pharmacological characteristics of this receptor have been described, for example, in the references by Humphries et al., Br. J. Pharmacology, (1994), 113, 1057-1063, and Fagura et al., Br. J. Pharmacology, (1998), 124, 157-164.
Compounds having antagonist activity at the P2YADP receptor are known, for example, from WO 98/28300, WO 97/03084, WO 94/18216 and EP-A-508 687 and are useful as anti-thrombotic agents in the treatment or prophylaxis of diseases such as unstable angina, coronary angioplasty (PTCA) and myocardial infarction.
It would be desirable to identify further compounds with binding activity at the P2YADP receptor, and also to identify further tissues/cell lines containing this receptor.
In accordance with the present invention, there is therefore provided a competition binding assay which comprises contacting a P2YADP receptor, preferably a human P2YADP receptor, with a P2YADP receptor radioligand and a candidate P2YADP receptor ligand, and measuring bound radioactivity.
The assay according to the present invention is very conveniently carried out on multi-well microtitre plates, thereby enabling a fast, simple and reproducible way of screening large numbers of potential P2YADP receptor ligands.
In the context of the present specification, the term xe2x80x9cP2YADP receptor ligandxe2x80x9d, unless otherwise indicated, defines a ligand, e.g. an agonist or antagonist, of the P2YADP receptor other than a naturally-occurring ligand. The ligand may, for example, be a chemical compound, or a salt or solvate thereof.
The radioligand used in the assay is a substance which binds to the P2YADP receptor and which may be synthesised containing one or more radioactive atoms. Examples of substances which when radiolabelled may be used as the radioligand include:
(a) Compounds of formula (I) 
xe2x80x83wherein
X is OH or NHR3;
R1 is C1-6-alkyl, C3-8-cycloalkyl or a phenyl group, each group being optionally substituted by one or more halogen atoms and/or OR4, NR4R5, C1-6-thioalkyl and/or C1-6-alkyl (itself optionally substituted by one or more halogen atoms); R2 is C1-8-alkyl or C2-8-alkenyl each of which is optionally substituted by one or more halogen atoms and/or OR4, NR4R5, C1-6-thioalkyl, C3-8-cycloalkyl, aryl and/or C1-6-alkyl groups; or R2 is a C3-8-cycloalkyl group optionally substituted by one or more halogen atoms and/or OR4, NR4R5, C1-6-thioalkyl, phenyl and/or C1-6-alkyl groups; the optional phenyl substituent being further optionally substituted by one or more halogen atoms and/or NO2, C(O)R4, OR4, NR4R5, C1-6-thioalkyl and/or C1-6-alkyl groups;
R3 is hydrogen or C1-6-alkyl substituted by one or more hydroxy and/or phenyl groups and optionally by one or more halogen atoms, wherein the phenyl group is substituted by one or more hydroxy groups and optionally substituted by one or more halogen atoms and/or NO2, C(O)R4, OR4, NR4R5, C1-6-thioalkyl and/or C1-6-alkyl groups, or R3 is a C1-6-alkyl group substituted by a C(O)NR4R5 or a COOH group and optionally by one or more halogen atoms and/or OR4, C(NH)NR4R5, C(O)NR4R5, phenyl and/or C1-6-alkyl groups, wherein the alkyl group is optionally substituted by one or more hydroxy and/or phenyl groups and wherein the phenyl group is optionally substituted as defined above for R3; or
R3 is a lactam ring of formula (i): 
xe2x80x83wherein Q is a (CH2)m moiety wherein m is 1, 2 or 3, Z is O, C(O) or CH2;
R4 and R5 each independently represent hydrogen, phenyl or a C1-6-alkyl wherein the alkyl group is optionally substituted by one or more phenyl groups; and salts and solvates thereof;
(b) Compounds of formula (II) 
xe2x80x83wherein
B is O or CH2;
X is selected from NR1R2, SR1 and C1-C7 alkyl;
Y is selected from NR1R2, SR1 and C1-C7 alkyl;
R1 and R2 is each and independently selected from H, or C1-C7 alkyl optionally substituted upon or within the alkyl chain by one or more of O, S, N or halogen;
R3 and R4 are both hydrogen, or R3 and R4 together form a bond;
A is COOH, C(O)NH(CH2)pCOOH, C(O)N[(CH2)qCOOH]2, C(O)NHCH(COOH)(CH2)rCOOH or 5-tetrazolyl, wherein p, q and r is each and independently 1, 2 or 3; and salts and solvates thereof;
(c) Compounds of formula (I) 
xe2x80x83wherein
R1 and R2 independently represent hydrogen or halogen;
R3 and R4 independently represent phenyl, or C1-C6 alkyl optionally substituted by one or more substituents selected from OR5, C1-C6 alkylthio, NR6R7, phenyl, COOR8 and halogen;
R5, R6, R7 and R8 independently represent hydrogen or C1-C6 alkyl;
X represents an acidic moiety; and salts and solvates thereof;
(d) Compounds of formula (IV) 
xe2x80x83wherein
Q represents CR1R2;
R represents O or CR3R4;
W represents O or CH2;
R1, R2, R3 and R4 independently represent hydrogen or halogen;
X represents S(O)nR5, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acylamino, CONR6R7, NR8R9, halogen, a 5- or 6-membered S containing heterocycle, or phenyl optionally substituted by C1-C6 alkyl;
n represents 0, 1 or 2;
R5 represents aryl or C1-C6 alkyl optionally substituted by one or more substituents selected from hydroxy, C1-C6 alkoxy, halogen and aryl;
R6, R7, R8 and R9 independently represent hydrogen or C1-C6 alkyl;
Y represents NH2 or C1-C6 alkoxy;
Z represents an acidic moiety;
in addition, when R represents CR3R4, then xe2x80x94Qxe2x80x94Z may also represent hydroxy or xe2x80x94OP(O)(OH)2, provided that:
i) when R is O, W is O, X is Cl, Y is NH2 and Z is xe2x80x94P(O)(OH)2, then CR1R2 does not represent CH2; and
ii) when R is O, W is O, X is SCH3, Y is NH2 and Z is xe2x80x94P(O)(OH)2, then xe2x80x94CR1R2 does not represent CH2, CF2 or CCl2; and salts and solvates thereof;
(e) Compounds of formula (V) 
xe2x80x83wherein
R1 is a C1-6 alkyl, C3-8-cycloalkyl or a phenyl group, each group being optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11 or C1-6 alkyl (itself optionally substituted by one or more halogen atoms);
R2 is C1-8 alkyl optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11, C3-8-cycloalkyl, aryl (optionally substituted by one or more alkyl groups and/or halogen atoms), or C1-6-alkyl; or R2 is a C3-8-cycloalkyl group optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11, C1-6-alkyl or phenyl (the latter two being optionally substituted by one or more substituents selected from halogen, NO2, C(O)R8, OR8, SR11, NR12R13, phenyl and C1-6-alkyl which is optionally substituted by one or more halogen atoms);
one of R3 or R4 is hydroxy and the other is hydrogen, hydroxy or NR9R10;
R5 is (CH2)nNR14R15 where n is 0 to 6 and R14 and R15 are independently hydrogen, C1-6-alkyl or phenyl; or R5 is CONR16R17 where R16 is hydrogen or C1-6-alkyl, and R17 is C1-6-alkyl or C3-6-cycloalkyl each of which is substituted by NR18R19 and optionally substituted by phenyl, or R17 is C1-6-alkyl or C3-6-cycloalkyl substituted by phenyl which is substituted by NR18R19 where R18 and R19 are independently hydrogen, C1-6-alkyl or phenyl; or R17 is a 5- to 8-membered saturated heterocycle containing one or more nitrogen atoms and optionally substituted on nitrogen by hydrogen, C1-6-alkyl or phenyl;
or R16 and R17 together with the nitrogen atom to which they are attached form a 5- to 8-membered ring which is substituted by NR18R19 as defined above; or
R16 together with R19 forms a 6- to 8-membered ring containing the two nitrogen atoms in which R17 and R18 are as defined above; or R5 is (CH2)pNR20CO(CH2)qOR21 or (CH2)pNR22(CH2)qNR23COR24 where p and q are independently 1 to 4 and R20, R21, R22 R23 and R24 are independently C1-4-alkyl or phenyl; or R5 is CHxe2x95x90CHCH2NR25R26 where R25 is hydrogen, C1-6 alkyl or phenyl and R26 is hydrogen or (CH2)yNR27R28 where y is 2-4 and R27 and R28 are independently hydrogen, C1-6 alkyl or phenyl;
R8, R9, R10 and R11 are independently hydrogen or C1-6-alkyl; and
R12 and R13 are independently hydrogen, C1-6-alkyl or acyl groups;
and salts and solvates thereof;
(f) Compounds of formula (VI) 
xe2x80x83wherein
R1 is a C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8-cycloalkyl or a phenyl group, each group being optionally substituted by one or more substituents selected from halogen, OR8 NR9R10, SR11 or C1-6 alkyl (itself optionally substituted by one or more halogen atoms); R2 is C1-8 alkyl optionally substituted by one or more substituents selected from halogen OR8, NR9R10, SR11, C3-8-cycloalkyl, aryl (optionally substituted by one or more alkyl groups and/or halogen atoms), or C1-6-alkyl; or R2 is a C3-8-cycloalkyl group optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11, C1-6-alkyl or phenyl, the latter two groups being optionally substituted by one or more substituents selected from halogen, NO2, C(O)R8, OR8, SR11, NR12R13, a fused 5- or 6-membered saturated ring containing one or two oxygen atoms, phenyl or C1-6-alkyl the latter two groups being optionally substituted by OR8, NR9R10 or one or more halogen atoms;
one of R3 and R4 is hydroxy and the other is hydrogen, hydroxy or NR9R10;
R is a group (CR5R6)mOR7 where m is 0 or 1, R5 and R6 are independently hydrogen, C1-6 alkyl or phenyl the latter two groups being optionally substituted by halogen, and R7 is hydrogen, C1-6 alkyl or (CR5R6)R14 where R5 and R6 are as defined above, n is 1 to 3 and, R14 is COOH, OR15, NR15R17 or CONR16R17;
or R is a C1-4 alkyl or C2-4 alkenyl group, each of which is substituted by one or more groups selected from xe2x95x90S, xe2x95x90O, xe2x95x90NR20 or OR21 and optionally substituted by one or more groups selected from halogen, C1-4 alkyl, phenyl, SR21, NO2 or NR22R23 (where R21, R22, and R23 are independently hydrogen, C1-4 alkyl or phenyl; R20 is OR24 or NR25R26, where R24 is hydrogen, C1-4 alkyl or phenyl, and R25 and R26 are independently hydrogen, C1-4 alkyl, aryl, C1-6 acyl, arylsulphonyl or arylcarbonyl);
R8 is hydrogen, C1-6 alkyl optionally substituted by halogen or R8 is phenyl optionally substituted by one or more substituents selected from halogen, NO2, C(O)R6, OR6, SR9, NR10OR11;
R9, R10 and R11 are independently hydrogen or C1-6 alkyl;
R12 and R13 are independently hydrogen, C1-6 alkyl, acyl, alkyl sulfonyl optionally substituted by halogen, or phenyl sulfonyl optionally substituted by C1-C4 alkyl; and R15, R16 and R17 are independently hydrogen or C1-6 alkyl; and salts and solvates thereof; and
(g) Compounds of formula (VII) 
xe2x80x83wherein
R1 is a C1-6 alkyl, C3-8-cycloalkyl or a phenyl group, each group being optionally substituted by one or more substituents selected from halogen, OR6, NR7R8, SR9 or C1-6 alkyl (itself optionally substituted by one or more halogen atoms);
R2 is C1-8 alkyl optionally substituted by one or more substituents selected from halogen, OR6, NR7R8, SR9, C3-8-cycloalkyl, aryl (optionally substituted by one or more alkyl groups and/or halogen atoms), or C1-6-alkyl; or R2 is a C3-8-cycloalkyl group optionally substituted by one or more substituents selected from halogen, OR6, NR7R8, SR9, C1-6-alkyl or phenyl (the latter two being optionally substituted by one or more substituents selected from halogen, NO2, C(O)R6, OR6, SR9, NR10R11, phenyl and C1-6-alkyl which is optionally substituted by one or more halogen atoms);
one of R3 or R4 is hydrogen and the other is hydroxy;
X is OH or NHR5;
R is a C1-6-alkyl group substituted by COOH or C(O)NR7R8 and optionally by one or more further substituents selected from halogen, OR12, C(NH)NR13R14, C(O)NR15R16, phenyl (optionally substituted by one or more groups selected from halogen, NO2, C(O)R6, OR20, NR7R8, SR9 and C1-6-alkyl) or C1-6-alkyl (optionally substituted by one or more hydroxy or phenyl groups);
or R5 is a lactam ring of formula (i): 
xe2x80x83wherein Q is a (CH2)m moiety where m is 1, 2 or 3, Z is O, C(O) or CH2 and R18 is hydrogen or C1-6-alkyl;
R6, R9, R12, R13, R14, R15 and R16 are independently hydrogen or C1-6-alkyl;
R7 and R8 are independently hydrogen, C1-6-alkyl (optionally substituted by one or more phenyl groups) or phenyl groups; and
R10 and R11 are independently hydrogen, C1-6-alkyl or acyl groups;
and salts and solvates thereof.
Compounds of formulae (I), (II), (III), (IV), (V), (VI) and (VII) are disclosed respectively in WO 98128300, WO 97/03084, WO 94/18216, EP-A-508 687, PCT/SE98/01392, PCT/SE98/01393 and PCT/SE98/01394 and the contents of these seven documents are incorporated herein by reference.
Examples of suitable salts that may be used include alkali metal (e.g. sodium or potassium), alkaline earth metal (e.g. calcium or magnesium), Group III metal (e.g. aluminium), ammonium, hydrochloride, hydrobromide, phosphate, acetate, fumarate, maleate, tartrate, citrate, oxalate, methanesulphonate and p-toluenesulphonate salts.
The techniques for radiolabelling substances may be those conventionally used in the art and therefore the radioligand may be prepared by methods known in the art.
The radioligand is most preferably a radiolabelled compound of formula (I) or(II) or a salt or solvate thereof, and is especially [125I]-[1S-[1xcex1,2xcex2,3xcex2,4xcex1(E)]]-2,3-dihydroxy-4-[7-3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylic acid, or [3H]-[1S-(1xcex1,2xcex2,3xcex2,4xcex1)]-4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoic acid, or a salt or solvate of any one thereof.
Advantageously, the radioligand has a specific activity of greater than 10 Ci/mmol and a P2YADP receptor activity (IC50) of less than 1 micromolar (xcexcM).
In the context of the present specification, xe2x80x9cspecific activityxe2x80x9d is defined as the activity per unit mass of substance containing a radioactive nuclide and is normally expressed as millicuries per milligram (mCi/mg) (kBq/mg), as millicuries per millimole (mCi/mmol) (kBq/mmol), or as curies per millimole (Ci/mmol) (GBq/mmol); and xe2x80x9cP2YADP receptor activity (IC50)xe2x80x9d is defined as the concentration, expressed in micromolar units, of radioligand required to inhibit the maximal aggregation response elicited by ADP according to the platelet aggregation assay as described in WO 98/28300. The platelet aggregation assay, which uses washed human platelets, is carried out in the following manner.
Human venous blood (100 ml) is divided equally between 3 tubes, each containing 3.2% trisodium citrate (4 ml) as anticoagulant. The tubes are centrifuged for 15 minutes at 240 G to obtain a platelet-rich plasma (PRP) to which 300 ng/ml prostacyclin is added to stabilize the platelets during the washing procedure. Red cell free PRP is obtained by centrifugation for 10 minutes at 125 G followed by further centrifugation for 15 minutes at 640 G. The supernatant is discarded and the platelet pellet resuspended in modified, Calcium Free Tyrode solution (10 ml) (CFT), composition: NaCl 137 mM, NaHCO3 11.9 mM, NaH2PO4 0.4 mM, KCl 2.7 mM, MgCl2 1.1 mM, dextrose 5.6 mM, gassed with 95% O2/5% CO2 and maintained at 37xc2x0 C. Following addition of a further 300 ng/ml PGI2, the pooled. suspension is centrifuged once more for 15 minutes at 640 G. The supernatant is discarded and the platelets are resuspended initially in 10 ml CFT with further CFT added to adjust the final platelet count to 2xc3x97105/ml. This final suspension is stored in a 60 ml syringe at 3xc2x0 C. with air excluded. To allow recovery from PGI2-inhibition of normal function, platelets are used in aggregation studies no sooner than 2 hours after final resuspension.
Aliquots of platelet suspension (3 ml) are added to tubes containing CaCl2 solution (60 xcexcl of 50 mM solution with a final concentration of 1 mM). Human fibrinogen (Sigma, F 4883) and 8-sulphophenyltheophylline (8-SPT, which is used to block any P1-agonist activity of test substance) are added to give final concentrations of 0.2 mg/ml (60 xcexcl of 10 mg/ml solution of clottable protein in saline) and 300 nM (10 xcexcl of 15 mM solution in 6% glucose), respectively. Platelets or buffer as appropriate are added in a volume of 150 xcexcl to the individual wells of a 96 well plate. All measurements are made in triplicate in platelets from each donor.
Aggregation responses in 96 well plates are measured using the change in absorbance given by the plate reader at 660 nm. Either a Bio-Tec Ceres 900C or a Dynatech MRX are used as the plate reader.
The absorbance of each well in the plate is read at 660 nm to establish a baseline figure. Saline or the appropriate solution of test substance (e.g. the radioligand) is added to each well in a volume of 10 gl to give a final concentration of 0, 0.01, 0.1, 1, 10 or 100 mM. The plate is then shaken for 5 minutes on an orbital shaker on setting 10 and the absorbance read at 660 nm. Aggregation at this point is indicative of agonist activity of the test substance. Saline or ADP (30 mM; 10 xcexcl of 450 mM) is then added to each well and the plate shaken for a further 5 minutes before reading the absorbance again at 660 nm. The concentration of test substance that produces a response which is half the maximum control ADP response is the IC50 value.
According to a preferred embodiment of the invention, the competition binding assay comprises
(i) isolating and washing human platelets or human platelet membranes,
(ii) incubating the platelets or platelet membranes with a P2YADP receptor radioligand and a candidate P2YADP receptor ligand,
(iii) filtering and washing the platelets or platelet membranes, and
(iv) measuring bound radioactivity.
In step (i) above, methods for isolating and washing human platelets or human platelet membranes are known in the art, e.g. as described by Connolly et al. (1992), J. Biol. Chem., 267, 6893-6898 and Biochim. et Biophys. Act. (1986), 854, 67-76.
In step (ii), the incubation is conveniently carried at a temperature in the range from 4 to 37xc2x0 C., for a period of time of from 5 to 120 minutes.
The present invention further provides the use of [125I]-[1S-[1xcex1,2xcex2,3xcex2,4xcex1(E)]]-2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylarnino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylic acid, or [3H]-[1S-(1xcex1,2xcex2,3xcex2,4xcex1)]-4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoic acid, or a salt or solvate of any one thereof, as a P2YADP receptor radioligand in a competition binding assay as hereinbefore defined.